Ecm hydrogel for treating esophageal inflammation

ABSTRACT

Methods are disclosed for inhibiting esophageal inflammation in a subject, that include administering to the esophagus of the subject with esophageal inflammation a therapeutically effective amount of an extracellular matrix (ECM) hydrogel. Methods are also disclosed for reducing esophageal stricture. Compositions are disclosed that include an esophageal extracellular matrix (ECM) hydrogel.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/465,985, filed Mar. 2, 2017, which is herein incorporated byreference in its entirety.

FIELD

This relates to the field of hydrogels, specifically to the use of anextracellular matrix (ECM) hydrogel for the treatment of esophagealinflammation such as Barrett's esophagus.

BACKGROUND

The incidence of esophageal adenocarcinoma (EAC) is rapidly rising;outpacing the rate of increase of all other cancers. EsophagealAdenocarcinoma (EAC) is associated with a dismal prognosis, with afive-year survival of less than 15%. The number of patients affected isup to 600% higher than in the 1970s' (Dubecz et al., J GastrointestSurg. 2013 Nov. 15; Prasad et al., Amer. J. Gastroentero.105(7):1490-502, 2010).

Barrett's esophagus involves metaplasia of the cells of the lower(distal) portion of the esophagus, and is characterized by thereplacement of the normal stratified squamous epithelium lining of theesophagus by simple columnar epithelium with goblet cells. Barrett'sesophagus is strongly associated with esophageal adenocarcinoma. Themain cause of Barrett's esophagus is thought to be an adaptation andresponse to chronic acid exposure from reflux esophagitis. The cells ofBarrett's esophagus, after biopsy, are classified into four generalcategories: nondysplastic, low-grade dysplasia, high-grade dysplasia,and frank carcinoma. High-grade dysplasia and early stages ofadenocarcinoma are usually treated by endoscopic resection andendoscopic therapies such as radiofrequency ablation, whilenondysplastic and low-grade patients are generally advised to undergoannual observation with endoscopy. A need remains for methods andcompositions that can be used for treating esophageal inflammation andBarrett's esophagus.

SUMMARY

Methods are disclosed for inhibiting inflammation and/or mitigating theeffects of esophageal inflammation in a subject. Methods are alsodisclosed for reducing stricture of the esophagus. These methods includeadministering to the esophagus of the subject, such as a subject withesophageal inflammation or a stricture or at risk for stricture, atherapeutically effective amount of an extracellular matrix (ECM)hydrogel, wherein the ECM hydrogel has the following characteristics: a)a time to 50% gelation of less than 30 minutes at a temperature of about37° C.; b) a flow viscosity suitable for infusion into the esophagus;and c) a stiffness of 10-600 Pascal (Pa), such as, but not limited to,10-70 Pa. In a specific non-limiting example, the hydrogel can be anesophageal ECM hydrogel. In another specific non-limiting example, thesubject can have Barrett's esophagus.

In additional embodiments, compositions are disclosed that include anesophageal extracellular matrix (ECM) hydrogel, wherein the esophagealECM hydrogel has the following characteristics: a) a time to 50%gelation of less than ten minutes at about 37° C.; b) a flow viscositysufficient for injection into the esophagus; and c) a stiffness of 10-70Pascal (Pa), wherein the composition is formulated for administration tothe esophagus. These compositions are of use in the methods disclosedherein.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Esophageal ECM Hydrogel Viscosity Profile.

FIG. 2. Esophageal ECM hydrogel stiffness.

FIG. 3. Esophageal ECM gelation time.

FIGS. 4A-4C. Viscosity profiles are tissue-specific.

FIGS. 5A-5C. Gel stiffness is tissue-specific.

FIGS. 6A-6C. ECM gelation time is tissue-specific.

FIG. 7. ECM hydrogels promote secretion of anti-inflammatory cytokines.

FIGS. 8A-8B. ECM promotes the chemotaxis of epithelial and stem cells.

FIG. 9. Dynamic reciprocity in clinical treatment of EAC.

FIGS. 10A-10B. Effects of hydrogel administration.

FIG. 11. Effects of hydrogel administration after 30 days.

FIGS. 12A-12B. Evaluation of safety of eECM hydrogel.

FIGS. 13A-13D. Histology.

FIG. 14. Use for treating stricture.

FIG. 15. Histological analysis, control dog.

FIG. 16. Histological analysis, dog treated with ECM hydrogel.

DETAILED DESCRIPTION

When manufactured as a hydrogel, extracellular-matrix (ECM) bioscaffoldshave bioactivity (Freytes et al., Biomaterials 29: 1630-7, 2008).However, ECM hydrogel has unique physical and mechanical properties.These distinctive properties extend the applications of ECM beyond thosepossible for the sheet form. The ECM hydrogel, in comparison to thesheet form, can be delivered to irregularly-shaped and sized surfaceareas, ensures contact of ECM with tissue that has an irregulartopology, does not require a fixation device (e.g. sutures, stent) anddoes not impart adverse rigidity to the area where it is implanted. Thehydrogel has the characteristic of being a liquid at room temperature(pre-gel), but becomes a gel when exposed to body temperature (37° C.),making it easily deliverable through devices like syringes, catheters,irrigators, and probes, among others.

ECM in sheet form has been used to treat late stage esophagealdysplastic and neoplastic disease, however, the need for a fixationdevice (i.e. stent), rigidity of the sheet form and relativeinvasiveness of implantation has not allowed it to expand its use. Thedistinctive properties of the ECM hydrogel make it ideal for applicationto a surface of the esophagus, and/or the treatment of esophagealdisease that is not treatable with the sheet form. The ECM hydrogel'sviscoelastic and mucoadhesion properties support its potential for thetreatment of esophageal disease. In addition, the hydrogel can be usedto reverse cancerous and pre-cancerous lesions in the esophagus.

Recently, the sheet form has been used for treatment of esophageal latestage cancerous and precancerous disease with success. For esophagealuse the sheet is placed circumferentially and held in place by a stent.There are limitations to the use of a sheet form such as the need forfixation devices (i.e. sutures or stents), its inability to fillirregularly sized defects and its limited area of coverage (to the sizeof the sheet). In the esophagus, these limitations have constrained theuse of the ECM sheet to the treatment of late stage esophageal disease,one of the few situations where the use of a temporary stent and anadvanced procedure is justified.

The disclosed hydrogels can be administered topically to the esophagus.The hydrogel can be administered to the lumen of the esophagus to coatthe surface. This is a non-invasive application. In some embodiments,the application is oral, such as by swallowing. In other embodiments,application can be gavage. The hydrogel is formed at the surface of theesophageal tissue. In some embodiments, the hydrogel coats the mucosa,and does not invade into the underlying submucosa or musculature.

It is disclosed herein that a hydrogel, such as a hydrogel produced fromesophageal ECM, can be used to treat Barrett's esophagus, and inhibitthe development of adenocarcinoma. The ECM hydrogel can inhibitinflammation and mitigate the effects of inflammation. The ECM hydrogelcan reduce stricture. In some embodiments, the ECM hydrogel is effectivewhen utilized at concentrations of about 2 mg/ml to about 20 mg/ml, suchas about 8 mg/ml to about 12 mg/ml.

Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Acid Protease: An enzyme that cleaves peptide bonds, wherein the enzymehas increased activity of cleaving peptide bonds in an acidic pH. Forexample and without limitation, acid proteases can include pepsin andtrypsin.

Barrett's Esophagus: An abnormal change (metaplasia or dysplasia) in thecells of the lower (distal) portion of the esophagus. Barrett'sesophagus is the diagnosis when the normal stratified squamousepithelium lining of the esophagus is replaced by simple columnarepithelium with goblet cells. Barrett's esophagus is found in 5-15% ofpatients who seek medical care for gastroesophageal reflux disease(GERD), although a large subgroup of patients with Barrett esophagus donot have symptoms. Barrett's esophagus is strongly associated withesophageal adenocarcinoma, and is considered to be a premalignantcondition. The main cause of Barrett's esophagus is thought to be anadaptation to chronic acid exposure from reflux esophagitis. The cellsof Barrett's esophagus, after biopsy, are classified into four generalcategories: non-dysplastic, low-grade dysplasia, high-grade dysplasia,and frank carcinoma.

Base: A compound or a solution of a compound with a pH greater than 7.For example and without limitation, the base is an alkaline hydroxide oran aqueous solution of an alkaline hydroxide. In certain embodiments,the base is NaOH or NaOH in PBS.

Comminute (comminution and comminuting): The process of reducing largerparticles into smaller particles, including, without limitation, bygrinding, blending, shredding, slicing, milling, cutting, shredding. ECMcan be comminuted while in any form, including, but not limited to,hydrated forms, frozen, air-dried, lyophilized, powdered, sheet-form.

Diagnosis: The process of identifying a disease by its signs, symptomsand results of various tests. The conclusion reached through thatprocess is also called “a diagnosis.” Forms of testing commonlyperformed include blood tests, medical imaging, and biopsy.

Extracellular Matrix (ECM): The non-cellular component of tissues andorgans. Natural ECMs (ECMs found in multicellular organisms, such asmammals and humans) are complex mixtures of structural andnon-structural biomolecules, including, but not limited to, collagens,elastins, laminins, glycosaminoglycans, proteoglycans, antimicrobials,chemoattractants, cytokines, and growth factors. in mammals, ECM oftencomprises about 90% collagen by dry weight mass, in its various forms.Biologic scaffolds can be created by removing the cells from a giventissue or organ. The composition and structure of ECMs vary depending onthe source of the tissue. For example, small intestine submucosa (SIS),urinary bladder matrix (UBM), esophagus (E) and liver stroma ECM eachdiffer in their overall structure and composition due to the uniquecellular niche needed for each tissue. An intact “extracellular matrix”and “intact ECM” bioscaffold consists of extracellular matrix thatideally retains activity of its structural and non-structuralbiomolecules, including, but not limited to, collagens, elastins,laminins, glycosaminoglycans, proteoglycans, antimicrobials,chemoattractants, cytokines, and growth factors, such as, withoutlimitation comminuted ECM as described herein.

The activity of the biomolecules within the ECM can be removedchemically or mechanically, for example, by chemical or enzymaticcross-linking and/or by dialyzing the ECM. Intact ECM essentially hasnot been enzymatically digested, cross-linked and/or dialyzed, meaningthat the ECM has not been subjected to a digestion, dialysis and/or across-linking; process, or conditions other than processes that occurnaturally during storage and handling of ECM prior to solubilization.Thus, ECM that is substantially cross-linked and/or dialyzed (inanything but a trivial manner which does not substantially affect thegelation and functional characteristics of the ECM in its uses describedherein) is not considered to be “intact.”

Esophagogastroduodenoscopy (EGD) or Upper Gastrointestinal Endoscopy: Adiagnostic endoscopic procedure that visualizes any upper part of thegastrointestinal tract up to the duodenum. An “esophageal endoscopy” isany endoscopic procedure that visualizes the esophagus. An esophagealendoscopy may sometimes be performed as part of an EGD or uppergastrointestinal endoscopy. The terms are not mutually exclusive unlessexpressly stated to be so.

Gelation: The formation of a gel from a sol.

Gastroesophageal Reflux Disease (GERD): A chronic symptom of mucosaldamage caused by stomach acid refluxing from the stomach into theesophagus. GERD is usually caused by changes in the barrier between thestomach and the esophagus, including abnormal relaxation of the loweresophageal sphincter, which normally holds the top (proximal portion) ofthe stomach closed, impaired expulsion of gastric reflux from theesophagus, or a hiatal hernia. These changes may be permanent ortemporary.

Flow Viscosity: A measure of the resistance of a fluid to gradualdeformation by shear stress or tensile stress. Viscosity is a propertyof a fluid which opposes the relative motion between the two surfaces ofthe fluid in a fluid that are moving at different velocities. When afluid is forced through a tube, particles that compose the fluidgenerally move more quickly near the tube's axis and more slowly nearits walls. Stress (such as a pressure difference between the two ends ofthe tube) is needed to overcome the friction between particle layers tokeep the fluid moving. For a given velocity pattern, the stress requiredis proportional to the fluid's viscosity. Viscosity is measured withviscometers and rheometers. Viscosity can be measured as pascal second(Pa*s). Water at 20° C. has a viscosity of 1.002 mPa*s.

Hydrogel: A network of polymer chains that are hydrophilic, sometimesfound as a colloidal gel in which water is the dispersion medium.Hydrogels are highly absorbent natural or synthetic polymeric networks.Hydrogels also possess a degree of flexibility similar to naturaltissue.

Inflammation: A localized response elicited by injury to tissue.Inflammation is characterized by the appearance in or migration into anytissue space, unit or region of any class of leukocyte in numbers thatexceed the number of such cells found within such region of tissue undernormal (healthy) circumstances. Inflammation is orchestrated by acomplex biological response of vascular tissues to harmful stimuli, suchas pathogens, damaged cells, or irritants.

Isotonic Buffered Solution: A solution that is buffered to a between 7.2and 7.8 and that has a balanced concentration of salts to promote anisotonic environment.

Low Grade Dysplasia and High Grade Dysplasia (of the Esophagus):Pathological conditions of the esophagus. Generally, in esophagealdysplasia there is an absence of apical mucin in the internal lining ofthe esophagus. Frequently, both an absence of goblet cells and mucindepletion in the non-goblet columnar cells are seen in dysplasticepithelium. At low power, these areas appear more hyperchromatic ascompared to uninvolved areas.

For high grade dysplasia, distortion of glandular architecture of theesophagus is usually present and may be marked; it is composed ofbranching and lateral budding of crypts, a villiform configuration ofthe mucosal surface, or intraglandular bridging of epithelium to form acribriform pattern of “back-to-back” glands. There is dysplasticepithelium on the mucosal surface with loss of nuclear polarity,characterized by “rounding up” of the nuclei, and absence of aconsistent relationship of nuclei to each other.

Preventing or treating: Inhibiting a disease refers to inhibiting thepartial or full development of a disease, for example in a person who isat risk for a disease such as one caused by inflammation. An example ofa person at risk for esophageal adenocarcinoma is someone with Barrett'sesophagus or GERD. Inhibiting a disease process includes preventing thedevelopment of the disease. “Treatment” refers to a therapeuticintervention that ameliorates a sign or symptom of a disease orpathological condition, such as after it has begun to develop. SheerStress: The component of stress coplanar with a material cross section.Shear stress arises from the force vector component parallel to thecross section. The formula to calculate average shear stress is forceper unit area

${\tau = \frac{F}{A}},$

where τ=the shear stress, F=the force applied, A=the cross-sectionalarea of material with area parallel to the applied force vector.

Stricture: A narrowing or tightening of the esophagus that causesswallowing difficulties. Symptoms of esophageal strictures includeheartburn, bitter or acid taste in the mouth, choking, coughing,shortness of breath, frequent burping or hiccups, pain or troubleswallowing, vomiting blood and/or weight loss. Stricture of theesophagus can be caused by gastroesophageal reflux disease, esophagitis,a dysfunctional lower esophageal sphincter, disordered motility, lyeingestion, or a hiatal hernia. Strictures can form after esophagealsurgery and other treatments such as laser therapy or photodynamictherapy. While the area heals, a scar forms, causing the tissue to pulland tighten, leading to difficulty in swallowing. Stricture can be aresult of inflammation. A barium swallow test or an uppergastrointestinal endoscopy can be used to diagnose esophageal stricture.

Stiffness: The rigidity of an object or fluid. The stiffness of theextracellular matrix is important for guiding the migration of cells indurotaxis. Stiffness can be measure in Pascal (Pa), which are one newtonper square meter.

Therapeutic agent: Used in a generic sense, it includes treating agents,prophylactic agents, and replacement agents. “Treatment” or “treating”means providing a substance, such as a ECM hydrogel, to a patient in anamount sufficient to measurably reduce, inhibit, or mitigate any diseasesymptom, slow disease progression, or cause disease regression. Incertain embodiments treatment of the disease may be commenced before thepatient presents symptoms of the disease. The disclosed methods inhibitesophageal inflammation and/or mitigate the effects of esophagealinflammation.

Therapeutically effective amount: A “therapeutically effective amount”of a composition, such as an ECM hydrogel, means an amount effective,when administered to a patient, to provide a therapeutic benefit such asan amelioration of symptoms, reduced decrease progression, or causedisease regression. A quantity of a specified ECM hydrogel is sufficientto achieve a desired effect in a subject being treated, such as toinhibit inflammation and/or mitigate the effects of such inflammation,such as stricture. A therapeutically effective amount can beadministered systemically or locally, such as to the esophagus. Inaddition, an effective amount of a ECM hydrogel can be administered in asingle dose, or in several doses over time. However, the effectiveamount will be dependent on the preparation applied, the subject beingtreated, the severity and type of the affliction, and the manner ofadministration of the compound. The ECM hydrogels of use in the methodsdisclosed herein have applications in both medical and veterinarysettings. Therefore, the general term “subject” or “patient” isunderstood to include all animals, including, but not limited to, humansor veterinary subjects, such as other primates, dogs, cats, horses, andcows.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”The term “about” indicates within 5 percent. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

Extracellular Matrix (ECM) Hydrogels

Methods of preparing ECM hydrogels, are disclosed for example, in U.S.Pat. No. 8,361,503. Any type of extracellular matrix tissue can be usedto produce a hydrogel which can be used in the methods as disclosedherein (see U.S. Pat. Nos. 4,902,508; 4,956,178; 5,281,422; 5,352,463;5,372,821; 5,554,389; 5,573,784; 5,645,860; 5,771,969; 5,753,267;5,762,966; 5,866,414; 6,099,567; 6,485,723; 6,576,265; 6,579,538;6,696,270; 6,783,776; 6,793,939; 6,849,273; 6,852,339; 6,861,074;6,887,495; 6,890,562; 6,890,563; 6,890.564; and 6,893,666 related toECM). In certain embodiments, the ECM is isolated from a vertebrateanimal, for example and without limitation, from a warm bloodedmammalian vertebrate animal including, but not limited to, humans,monkeys, horses, pigs, cows and sheep. In specific non-limitingexamples, the ECM is porcine or human.

The ECM can be derived from any organ or tissue, including withoutlimitation, urinary bladder, intestine, liver, esophagus and dermis. Inone embodiment, the ECM is isolated from a urinary bladder. In anotherembodiment, the ECM is from an esophagus. The ECM may or may not includethe basement membrane portion of the ECM. In certain embodiments, theECM includes at least a portion of the basement membrane. In otherembodiments, the ECM is harvested from a cell culture. The ECM hydrogelcan be produced by a combination of two or more tissue sources.

As disclosed in U.S. Pat. No. 8,361,503 (incorporated herein byreference), a urinary bladder ECM, such as porcine bladder ECM isprepared by abrading bladder tissue to remove the outer (abluminal)layers including both the tunica serosa and the tunica muscularis usinga longitudinal wiping motion with a scalpel handle and moistened gauze.Following eversion of the tissue segment, the luminal portion of thetunica mucosa is delaminated from the underlying tissue using the samewiping motion. In some embodiments, perforation of the submucosa isprevented. After these tissues are removed, the resulting ECM consistsmainly of the tunica submucosa. The production of hydrogels fromdecellularized dermal ECM is disclosed in Wolf et al., Biomaterials 33:7028-7038, 2012, incorporated herein by reference. The production of ECMfrom esophageal tissue is disclosed, for example, in Badylak et al. JPediatr Surg. 35(7):1097-103, 2000 and Badylak et al., J Surg Res. 2005September; 128(1):87-97, 2005, both incorporated herein by reference.U.S. Pat. No. 6,893,666, incorporated herein by reference, disclosesproduction of ECM from urinary bladder, skin, esophagus and smallintestine.

Commercially available ECM preparations can also be used in the methods,devices and compositions described herein. In one embodiment, the ECM isderived from small intestinal submucosa or SIS. Commercially availablepreparations include, but are not limited to, SURGISIS™, SURGISIS-ES™,STRATASIS™, and STRATASIS-ES™ (Cook Urological Inc.; Indianapolis, Ind.)and GRAFTPATCH™ (Organogenesis Inc.; Canton, Mass.). In anotherembodiment, the ECM is derived from dermis. Commercially availablepreparations include, but are not limited to PELVICOL™ (sold asPERMACOL™ in Europe; Bard, Covington, Ga.), REPLIFORM™ (Microvasive;Boston, Mass.) and ALLODERM™ (LifeCell; Branchburg, N.J.). In anotherembodiment, the ECM is derived from urinary bladder. Commerciallyavailable preparations include, but are not limited to UBM (AcellCorporation; Jessup, Md.).

Source tissue used for preparation of ECM can be harvested in a largevariety of ways and once harvested, a variety of portions of theharvested tissue may be used. ECM has also been prepared from theesophagus and small intestine, and hydrogels have been prepared fromthis ECM, see, for example, Keane et al., Tissue Eng. Part A, 21(17-18):2293-2300, 2015, incorporated herein by reference. Esophageal ECM can beprepared by mechanically separating the mucosa and submucosa from themuscularis externa and digesting the mucosal layers in a bufferincluding trypsin, followed by exposure to sucrose, TRITON-X100®,deoxycholic acid, peracetic acid and DNAse. Small intestinal submucosa(SIS) can be prepared by mechanically removing the superficial layers ofthe tunica mucosa, tunica serosa, and tunica muscularis externa from theintact small intestine, leaving the submucosa, muscularis mucosa, andbasilar stratum compactum intact. The SIS is then treated with peraceticacid. Exemplary protocols are provided in Keane et al.

In one embodiment, the ECM is isolated from harvested porcine urinarybladder to prepare urinary bladder matrix (UBM). Excess connectivetissue and residual urine are removed from the urinary bladder. Thetunica serosa, tunica muscularis externa, tunica submucosa and most ofthe muscularis mucosa can be removed by mechanical abrasion or by acombination of enzymatic treatment, hydration, and abrasion. Mechanicalremoval of these tissues can be accomplished by abrasion using alongitudinal wiping motion to remove the outer layers (particularly theabluminal smooth muscle layers) and even the luminal portions of thetunica mucosa (epithelial layers). Mechanical removal of these tissuesis accomplished by removal of mesenteric tissues with, for example,Adson-Brown forceps and Metzenbaum scissors and wiping away the tunicamuscularis and tunica submucosa using a longitudinal wiping motion witha scalpel handle or other rigid object wrapped in moistened gauze. Theepithelial cells of the tunica mucosa can also be dissociated by soakingthe tissue in a de-epithetializing solution, for example and withoutlimitation, hypertonic saline. The resulting UBM comprises basementmembrane of the tunica mucosa and the adjacent tunica propria, which isfurther treated with peracetic acid, lyophilized and powdered, see U.S.Pat. No. 8,361,503.

In some embodiments, the epithelial cells can be delaminated first byfirst soaking the tissue in a de-epithelializing solution such ashypertonic saline, for example and without limitation, 1.0 N saline, forperiods of time ranging from 10 minutes to 4 hours. Exposure tohypertonic saline solution effectively removes the epithelial cells fromthe underlying basement membrane. The tissue remaining after the initialdelamination procedure includes epithelial basement membrane and thetissue layers abluminal to the epithelial basement membrane. This tissueis next subjected to further treatment to remove the majority ofabluminal tissues but not the epithelial basement membrane. The outerserosal, adventitial, smooth muscle tissues, tunica submucosa and mostof the muscularis mucosa are removed from the remainingde-epithelialized tissue by mechanical abrasion or by a combination ofenzymatic treatment, hydration, and abrasion.

ECM can be sterilized by any number of standard techniques, including,but not limited to, exposure to peracetic acid, low dose gammaradiation, gas plasma sterilization, ethylene oxide treatment,supercritical CO₂, or electron beam treatment. More typically,sterilization of ECM is obtained by soaking in 0.1% (v/v) peraceticacid, 4% (v/v) ethanol, and 95.9% (v/v) sterile water for two hours. Theperacetic acid residue is removed by washing twice for 15 minutes withPBS (pH=7.4) and twice for 15 minutes with sterile water. ECM materialcan be sterilized by propylene oxide or ethylene oxide treatment, gammairradiation treatment (0.05 to 4 mRad), gas plasma sterilization,peracetic acid sterilization, supercritical CO₂, or electron beamtreatment. The ECM can also be sterilized by treatment withglutaraldehyde, which causes cross linking of the protein material, butthis treatment substantially alters the material such that it is slowlyresorbed or not resorbed at all and incites a different type of hostremodeling which more closely resembles scar tissue formation orencapsulation rather than constructive remodeling. Cross-linking of theprotein material can also be induced with carbodiimide or dehydrothermalor photooxidation methods. As disclosed in U.S. Pat. No. 8,361,503, ECMis disinfected by immersion in 0.1% (v/v) peracetic acid (a), 4% (v/v)ethanol, and 96% (v/v) sterile water for 2 h. The ECM material is thenwashed twice for 15 min with PBS (pH=7.4) and twice for 15 min withdeionized water.

Following isolation of the tissue of interest, decellularization isperformed by various methods, for example and without limitation,exposure to hypertonic saline, peracetic acid, TRITON-X® or otherdetergents. Sterilization and decellularization can be simultaneous. Forexample and without limitation, sterilization with peracetic acid,described above, also can serve to decellularize the ECM. DecellularizedECM can then be dried, either lyophilized (freeze-dried) or air dried.Dried ECM can be comminuted by methods including, but not limited to,tearing, milling, cutting, grinding, and shearing. The comminuted ECMcan also be further processed into a powdered form by methods, forexample and without limitation, such as grinding or milling in a frozenor freeze-dried state. In order to prepare solubilized ECM tissue,comminuted ECM is digested with an acid protease in an acidic solutionto form a digest solution.

The digest solution of ECM typically is kept at a constant stir for acertain amount of time at room temperature. The ECM digest can be usedimmediately or be stored at −20° C. or frozen at, for example andwithout limitation, −20° C. or −80° C.

Once the ECM is solubilized (typically substantially completely) the pHof the solution is raised to between 7.2 and 7.8, and according to oneembodiment, to pH 7.4. Bases, such as bases containing hydroxyl ions,including NaOH, can be used to raise the pH of the solution. Likewisebuffers, such as an isotonic buffer, including, without limitation,Phosphate Buffered Saline (PBS), can be used to bring the solution to atarget pH, or to aid in maintaining the pH and ionic strength of the gelto target levels, such as physiological pH and ionic conditions. Thisforms a “pre-gel” solution. The neutralized digest solution (pre-gel)can be gelled at temperatures approaching 37° C., wherein thetemperature approaches physiological temperature. The method typicallydoes not include a dialysis step prior to gelation, yielding amore-complete ECM-like matrix that typically gels at 37° C. at specificrates (see below).

Thus, the ECM typically can be derived from mammalian tissue, such as,without limitation from one of urinary bladder, esophagus, or smallintestine. The ECM hydrogel can be produced from two or more tissuesource, such as 2, 3, or 4 tissue sources. In one non-limitingembodiment, the ECM is lyophilized and comminuted. The ECM is thensolubilized with an acid protease in an acidic solution to producedigested ECM, such as esophageal ECM. The acid protease may be, withoutlimitation, pepsin or trypsin, or a combination thereof. The ECM canthen be solubilized at an acid pH suitable or optimal for the protease,such as greater than about pH 2, or between pH and 4, for example in a0.01M HCl solution. The solution typically is solubilized for about 12to about 48 hours, depending upon the tissue type (e.g., see examplesbelow), with mixing (stirring, agitation, admixing, blending, rotating,tilting, etc.). ECM hydrogel is prepared by (i) comminuting anextracellular matrix, (ii) solubilizing intact, non-dialyzed ornon-cross-linked extracellular matrix by digestion with an acid proteasein an acidic solution to produce a digest solution, (iii) raising the pHof the digest solution to a pH between 7.2 and 7.8 to produce aneutralized digest solution (pre-gel solution), and (iv) gelling thesolution at a temperature of approximately 37° C. within the esophagusof a subject of interest.

The ECM hydrogel, when exposed to temperatures of about 37° C., formsthe gel. The ECM hydrogel in the “pre-gel” form can be frozen and storedat, for example and without limitation, −20° C. or −80° C. The ECMhydrogel in the “pre-gel” form can be stored at room temperature, suchabout 25° C. Thus, the ECM hydrogel is in the pre-gel form at below 37°C., such as at 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4° C. The ECM hydrogel can be frozen for storage,and thus, can be stored at below 0° C. As used herein, the term “pre-gelform” or “pre-gel” refers to the ECM hydrogel wherein the pH isincreased, but has not gelled. For example and without limitation, anECM hydrogel in the pre-gel form has a pH between 7.2 and 7.8. The ECMhydrogel can be delivered in a pre-gel form to a subject with esophagealinflammation, such as orally, via a catheter, or endoscopically.

The ECM hydrogel in the pre-gel form s amenable to introduction into theesophagus of a patient. Once introduced into the esophagus, which isapproximately 37° C., the ECM hydrogel gels and coats the esophagus.Without being bound by theory, the ECM hydrogel includes many nativesoluble factors. such as, but not limited to, cytokines. The specificcharacteristics of non-dialyzed (whole ECM preparations prepared from avariety of tissues, such as the esophagus, are disclosed herein. Thehydrogel gels with kinetics such that the ECM hydrogel can beadministered via oral administration, endoscopic administration, or viaa catheter into the esophagus, and the hydrogel subsequently gelswithin, and coats, the esophagus.

In some embodiments, the ECM hydrogel has the following characteristics:a) a time to 50% gelation of less than 30 minutes at a temperature ofabout 37° C.; b) a flow viscosity suitable for infusion into theesophagus; and c) a stiffness of i) about 10 to about 300 Pascal (Pa),ii) about 10 to about 450 Pa; iii) about 10 to about 600 Pa, iv) about 5to about 1,000 Pa, v) about 10 to 1,000 Pa, or vi) about 10 to about 70Pa.

In embodiments, the ECM hydrogel has the following characteristics: a) atime to 50% gelation of less than 30 minutes at a temperature of about37° C.; b) a flow viscosity suitable for infusion into the esophagus;and c) a stiffness of about 10 to about-300 Pascal (Pa). In otherembodiments, the ECM hydrogel has the following characteristics: a) atime to 50% gelation of less than 30 minutes at a temperature of about37° C.; b) a flow viscosity suitable for infusion into the esophagus;and c) a stiffness of about 10 to about 450 Pascal (Pa). In otherembodiments, the ECM hydrogel has the following characteristics: a) atime to 50% gelation of less than 30 minutes at a temperature of about37° C.; b) a flow viscosity suitable for infusion into the esophagus;and c) a stiffness of about 10 to about 600 Pascal (Pa).

In other embodiments, the ECM hydrogel has the followingcharacteristics: a) a time to 50% gelation of less than 30 minutes at atemperature of about 37° C.; b) a flow viscosity suitable for infusioninto the esophagus; and c) a stiffness of about 5 to about 1,000 Pascal(Pa). In other embodiments, the ECM hydrogel has the followingcharacteristics: a) a time to 50% gelation of less than 30 minutes at atemperature of about 37° C.; b) a flow viscosity suitable for infusioninto the esophagus; and c) a stiffness of about 10 to about 1,000 Pascal(Pa). In more embodiments, the ECM hydrogel has the followingcharacteristics: a) a time to 50% gelation of less than 30 minutes at atemperature of about 37° C.; b) a flow viscosity suitable for infusioninto the esophagus; and c) a stiffness of 10-70 Pascal (Pa).

In specific non-limiting examples, the ECM hydrogel is an esophagealhydrogel. In other specific non-limiting examples, the ECM hydrogel canbe produced from two or more tissue sources. In further non-limitingexamples, the ECM hydrogel can be produced from urinary bladder or smallintestine.

In additional specific non-limiting examples, the ECM hydrogel isproduced by (a) solubilizing acellular extracellular matrix (ECM) bydigestion of tissue with an acid protease in an acidic solution toproduce digested esophageal ECM; (b) raising the pH of the digested ECMto a pH between 7.2 and 7.8 to produce a neutralized digest solution;(c) diluting the digested ECM to a concentration of about 2 mg/ml toabout 16 mg/ml, such as about 8 mg/ml to about 12 mg/ml of the ECMhydrogel. This hydrogel is then introduced into the esophagus of thesubject, wherein it gels. The ECM can be esophageal ECM.

The ECM hydrogels of use in the methods disclosed herein have a time to50% gelation of less than 30 minutes at a temperature of about 37° C.,such as less than 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17,16, 15, 14, 13, 12, 1, 10, 9, 8, 7, 6, 5, 4, 3 minutes. In someembodiments, the ECM hydrogels have a time to gelation of less than 10minutes at a temperature of about 37° C. In other embodiments, the timeto 50% gelation is about 3 to about 30 minutes at a temperature of about37° C. In further embodiments, the time to 50% gelation is about 4 toabout 10 minutes at a temperature of about 37° C. In yet otherembodiments the time to 50% gelation is about 5 to about 10 minutes orabout 10 to about 20 minutes at a temperature of about 37° C.

The disclosed ECM hydrogels can have a flow viscosity suitable forinfusion into the esophagus. In some embodiments, the ECM hydrogel has aflow viscosity of about 10 to about 100 Pa*s at a sheer rate of 0.2/s,such as about 10, 20, 30, 40, 50, 60, 70, 80, or 90 Pa*s at a sheer rateof 0.2/s. In further embodiments, the ECM hydrogel has a flow viscosityof about 1 to about 40 Pa*s at a shear rate of 0.1/s, such as about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40 Pa*s at a shearrate of 0.1/s.

In other embodiments, the ECM hydrogel has a flow viscosity of about0.01 to about 0.20 Pa*s at a sheer rate of 1000/s, or of about 0.01 toabout 0.10 Pa*s at a sheer rate of 1000/s, such as about 0.01, 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14,0.15, 0.16, 0.17, 0.19 or 0.2 at a sheer rate of 1000/s.

In more embodiment, the ECM hydrogel has about 0.02 to about 0.8 Pa*s ata shear rate of 100/s, or of about 0.1 to about 0.8 Pa*s at a shear rateof 100/s, such as about 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09,0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.08 Pa*s.

In further embodiments, the ECM hydrogel has a flow viscosity of about10 to about 100 Pa*s at a sheer rate of 0.2/s and a flow viscosity ofabout 0.01 to about 0.10 Pa*s at a sheer rate of 1000/s. In moreembodiments, the ECM hydrogel has a flow viscosity of 1 to 40 Pa*s at ashear rate of 0.1/s and 0.01 to 0.2 Pa*s at a shear rate of 1000/s.

In other embodiments, the ECM hydrogel has a flow viscosity of about 1to about 40 Pa*s, such as 1 to about 30 Pa*s, or 1 to about 20 Pa*s, or1 to about 10 Pa*s at a sheer rate of 1/s, such as about 1, 2, 3, 4, 5,6, 7, 8, or 9 Pa*s at a sheer rate of 1/s. The shear rate can be, forexample, 10, 20, 30 or 40 Pa*s at a sheer rate of 1/s. In otherembodiments, the ECM hydrogel has a flow viscosity of about 0.05 toabout 0.20 at a sheer rate of 100/s, such as about 0.05, 0.06, 0.07,0.08, 0.09, 0.1, 0.15 or 0.2 at a sheer rate of 100/s. the flowviscosity is about 0.1 to about 25 Pa*s at a shear rate of 1/s, and isabout 0.02 to about 0.8 Pa*s at a shear rate of 100/s. In additionalembodiments, the flow viscosity is about 1 to about 10 Pa*s at a shearrate of 1/s, and is about 0.05 to about 0.20 at a shear rate of 100/s.

In further embodiments, the ECM hydrogel has a flow viscosity of about10 to about 100 Pa*s at a sheer rate of 0.2/s. In other embodiments, theECM hydrogel has a flow viscosity of about 0.01 to about 0.10 Pa*s at asheer rate of 1000/s. In other embodiments, the ECM hydrogel has a flowviscosity of about 1 to about 40 Pa*s at a shear rate of 0.1/s and is0.01 to 0.2 Pa*s at a shear rate of 1000/s.

The disclosed ECM hydrogels have a stiffness i) about 10 to about 300Pascal (Pa), ii) about 10 to about 600 Pa, iii) about 5 to about 1,000Pa, iv) about 10 to 1,000 Pa, or v) about 10 to about 70 Pa. The ECMhydrogel can have a stiffness of about 10 to about 300 Pascal (Pa), suchas about 10 to about 70 Pa, about 10 to about 100 Pascal (Pa), or about10 to about 150 Pa, about 10 to about 200 Pa, or about 10 to about 250Pa. In some embodiments, the disclosed ECM hydrogels have a stiffness ofabout 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 Pa. In otherembodiments, the disclosed ECM hydrogels have a stiffness of about 10 toabout 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 Pa. In furtherembodiments, the disclosed ECM hydrogel can have a stiffness of about70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 Pa.

In some embodiments, the ECM concentration in the hydrogel is about 2mg/ml to about 20 mg/ml, such as about 8 mg/ml to about 12 mg/ml orabout 2 mg/ml to about 16 mg/ml. In other embodiments, the ECMconcentration in the hydrogel is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or 16 mg/ml. Exemplary concentrations of use include,but are not limited to, about 9 mg/ml to about 11 mg/ml, and about 10/mgto about 12 mg/ml. Additional exemplary concentrations include about 8mg/ml to about 10 mg/ml, about 8 mg/ml to about 11 mg/ml, about 8 mg/mlo about 13 mg/ml, about 8 mg/ml to about 14 mg/ml, about 8 mg/ml toabout 15 mg/ml, and about 8 mg/ml to about 16 mg/ml. Further exemplaryconcentrations of use also include about 6 mg/ml to about 12 mg/ml,about 13 mg/ml, about 14 mg/ml, about 15 mg/ml or about 16 mg/ml.

The disclosed ECM hydrogels can be provided as components of a kit. TheECM hydrogel can be provided in frozen or lyophilized form. In someembodiments, the kit can include the components needed to form thehydrogel, such as one container including the hydrogel, such as in alyophilized form, one container including a solution for solubilizingthe lyophilized hydrogel, and optionally a container comprising aneutralizing solution for neutralizing the solubilized form. In otherembodiments, the kit can include a container including the solubilizedhydrogel, and a second container including a neutralizing agent.

Optionally, such a kit includes additional components includingpackaging, instructions and various other reagents, such as buffers,substrates, or other therapeutic ingredients. The kit can include acontainer and a label or package insert on or associated with thecontainer. Suitable containers include, for example, bottles, vials,syringes, etc. The containers may be formed from a variety of materialssuch as glass or plastic. The container typically holds a compositionincluding the ECM hydrogel, such as in frozen or lyophilized form, whichis effective for inhibiting esophageal inflammation and/or mitigatingthe effects of esophageal inflammation in a subject. In severalembodiments, the container may have a sterile access port (for examplethe container may be an intravenous solution bag or a vial having astopper pierceable by a hypodermic injection needle). The label orpackage insert indicates that the composition is used for treating theparticular condition, such as Barrett's esophagus.

The label or package insert typically will further include instructionsfor use. The package insert typically includes instructions customarilyincluded in commercial packages of therapeutic products that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts. The instructional materials may be written, in an electronicform (such as a computer diskette or compact disk) or may be visual(such as video files). The kits may also include additional componentsto facilitate the particular application for which the kit is designed,such as needles or catheters. The kits may additionally include buffersand other reagents routinely used for the practice of a particularmethod. Kits and appropriate contents are well known to those of skillin the art.

Methods of Treatment

Methods are disclosed herein for treating esophageal inflammation.Methods are also disclosed for reducing stricture of the esophagus.Without being bound by theory, an ECM hydrogel allows treatment of earlystage neoplastic esophageal disease and dysplastic esophageal disease.The disclosed hydrogels can be used to treat both pre-neoplastic andneoplastic esophageal diseases. The versatility of the disclosedhydrogels allow them to be used to treat esophageal burns, ulcerationsand other pathologies which can extend throughout multiple,non-connected areas of the esophagus. The disclosed hydrogels are alsoof use for treatment of long segment or disperse esophageal injurieswithout the need for the use of stent or invasive techniques. It is notfeasible to use the sheet form of ECM in these conditions, becausemultiple stents and ECM sheets across the surface of the esophagus wouldbe required. Furthermore, the sheet and hydrogel form of ECM have majordifferences in terms of active components. Any of the disclosedhydrogels are of use in these methods of treatment. One of skill in theart, such as a skilled physician, can readily identify therapeuticefficacy.

Thus, a subject can be selected that has esophageal inflammation. Asubject can has be selected that has, or is at risk for having structureof the esophagus.

In some embodiments, the subject is apparently healthy, such as asubject who does not exhibit symptoms of esophageal adenocarcinoma (EAC)(for example, does not have EAC, and/or has not previously hadgastroesphogeal reflux disease (GERD) or Barrett's esophagus), but hasesophageal inflammation. In some examples, a healthy subject is one thatif examined by a medical professional, would be characterized as healthyand free of symptoms, such as GERD. However, the subject has esophagealinflammation, for example as determined by endoscopic administration. Insome embodiments, the disclosed methods inhibit this inflammation.

In other embodiments, the subject has GERD and/or Barrett's esophagus.In specific non-limiting examples, the subject can use acid reducingdrugs such as proton pump inhibitors or histamine antagonists tosuppress gastroesophageal discomfort. The subject may be at increasedrisk due to smoking and/or alcohol use. The subject can have low gradedysplasia or high grade dysplasia of the esophagus. In some embodiments,the subject does not have esophageal adenocarcinoma. However, thesubject can be at risk for esophageal adenocarcinoma.

In some embodiments, the method inhibits or reverses the development ofesophageal neoplasia in the subject. In other embodiments, the methodrestores the epithelial barrier in the esophagus of the subject. Infurther embodiments, the method increases chemotaxis of both epithelialcells and/or stem cells to the site of injury in the esophagus of thesubject. In further embodiments, the method inhibits the development ofesophageal adenocarcinoma. In other embodiments the treatment permitsthe subject to reduce or avoid use of proton pump inhibitors and/orhistamine antagonist drugs. However, the disclosed methods can be usedin conjunction with proton pump inhibitors and/or histamine antagonistdrugs.

In further embodiments, treatment reduces stricture as compared to acontrol, such as a subject not treated with the ECM hydrogel. Thetreatment can increase circumference of the esophagus as compared to acontrol, such as a subject not treated with the ECM hydrogel.

An ECM hydrogel, as disclosed herein, is maintained at a temperature ator below which it gels, such as at or below room temperature (e.g.,about 25° C.). The ECM hydrogel can be maintained, for example, at 25°C. or 4° C. prior to administration. An effective amount of the ECMhydrogel, in the pre-gel form, is then administered to the esophagus ofthe subject. The ECM hydrogel can be administered orally, such that thehydrogel is swallowed by the subject and gels when delivered to theesophagus. The ECM hydrogel can be administered directly to theesophagus, using either a catheter or by endoscopic administration. TheECM hydrogel gels in the esophagus of the subject, which is at atemperature of approximately 37° C. In some embodiments, about 5 toabout 60 ml of the ECM hydrogel is administered to the subject, such asabout 10 ml to about 30 ml of the ECM hydrogel, such as about 10, 15,20, 25 or 20 ml of the ECM hydrogel. The ECM hydrogel can be provided inin a lyophilized or frozen form, and reconstituted just prior toadministration to the subject.

The disclosed methods include administering to the esophagus of thesubject, such as, but not limited to, a subject with esophagealinflammation, or a subject with stricture or at risk for structure, atherapeutically effective amount of an ECM hydrogel as disclosed herein,in the pre-gel form, and allowing the hydrogel to gel in the esophagusof the subject. In some embodiments, the ECM hydrogel a) a time to 50%gelation of less than 30 minutes at a temperature of about 37° C., b) aflow viscosity suitable for infusion into the esophagus; and c) astiffness of 10-70 Pascal (Pa). However, any of the hydrogels disclosedabove can be utilized. The ECM hydrogel can, in some embodiments, bederived from any mammalian tissue, such as but not limited to porcine orhuman tissue, and be, in some non-limiting examples, urinary bladder,small intestine, or the esophagus. Any of the hydrogels disclosed abovecan be used in for the treatment of esophageal inflammation and/or forrestoring an epithelial barrier in the esophagus of a subject. Any ofthe hydrogels disclosed above can also be used to treat stricture. Insome embodiments, local delivery to the surface avoids any undesiredside effects. In specific non-limiting example, the subject hasBarrett's esophagus or is at risk for having Barrett's esophagus.

The disclosed hydrogels can be administered topically to the mucosa ofthe esophagus in the pre-gel form. The hydrogel can be administered tothe lumen of the esophagus to coat the surface, using a non-invasivemethod of application. In some embodiments, the application is oral,such as by swallowing the hydrogel in the pre-gel form. In otherembodiments, application can be gavage, wherein the hydrogel in thepre-gel form is place at the desired location. In some embodiments, thehydrogel coats the mucosa, and does not invade into the underlyingsubmucosa or musculature.

One of skill in the art can readily formulate the hydrogel, such thatthe pre-gel form can be swallowed by the subject being treated. In yetanother embodiment, the hydrogel is provided in the pre-gel. form viaendoscopy to make sure that the medicament is locally delivered, such asspecifically to the region of the esophagus that needs treatment. Forexample, local delivery of the hydrogel can be via anendoscope/gastroscope. Generally, delivery is topical to the mucosa ofthe esophagus, in order to non-invasively deliver the pre-gel form ofthe hydrogel. The hydrogel gels at the surface and coats the desiredarea of the mucosa. In some embodiments, The ECM hydrogel gels andprovides a protective barrier to protect the mucosa.

In some embodiments, esophagogastroduodenoscopy (EGD) or uppergastrointestinal endoscopy can be performed for the subject of interest.These procedures can be performed before the application of thehydrogel, select the subject of interest. These procedures can also beperformed following the use of the disclosed methods, to evaluate theeffects on the subject, and to determine if additional applications arenecessary.

Exemplary Embodiments

Clause 1. A method for inhibiting esophageal inflammation or reducingesophageal stricture in a subject, comprising administering to theesophagus of the subject with esophageal inflammation a therapeuticallyeffective amount of an extracellular matrix (ECM) hydrogel, wherein theECM hydrogel has the following characteristics: a) a time to 50%gelation of less than 30 minutes at a temperature of about 37° C.; b) aflow viscosity suitable for infusion into the esophagus; and c) astiffness of i) about 10 to about 300 Pascal (Pa), ii) about 10 to about450 Pa; iii) about 10 to about 600 Pa, iv) about 5 to about 1,000 Pa, v)about 10 to 1,000 Pa, or vi) about 10 to about 70 Pa, thereby inhibitingesophageal inflammation or reducing esophageal stricture in the subject.

Clause 2. The method of Clause 1, wherein the time to 50% gelation isabout 3 to about 30 minutes at about 37° C.

Clause 3. The method of Clause 1, wherein the time to 50% gelation isabout 3 to about 10 minutes at about 37° C.

Clause 4. The method of Clause 2, wherein the time to 50% gelation isabout 4 to about 10 minutes.

Clause 5. The method of any one of Clauses 1-4, wherein the flowviscosity is about 1 to about 40 Pa*s at a shear rate of about 0.1/s andis about 0.01 to about 0.2 Pa*s at a shear rate of 1000/s.

Clause 6. The method of any one of Clauses 1-4, wherein the flowviscosity is about 0.1 to about 25 Pa*s at a shear rate of 1/s, and isabout 0.02 to about 0.8 Pa*s at a shear rate of about 100/s.

Clause 7. The method of any one of Clauses 1-6, wherein the ECM hydrogelhas a stiffness of 10-70 Pa.

Clause 8. The method of any one of Clauses 1-7, wherein the ECM hydrogelis an esophageal ECM hydrogel.

Clause 9. The method of any one of Clauses 1-8, wherein the ECMconcentration in the hydrogel is 2 mg/ml to about 16 mg/ml.

Clause 10. The method of any one of Clauses 1-9, wherein the ECMhydrogel is administered orally, endoscopically or via a catheter.

Clause 11. The method of any one of Clauses 1-10, wherein the ECMhydrogel is produced by (a) solubilizing decellularized extracellularmatrix (ECM) by digestion of tissue with an acid protease in an acidicsolution to produce digested esophageal ECM; and (b) raising the pH ofthe digested esophageal ECM to a pH between 7.2 and 7.8 to produce aneutralized digest solution.

Clause 12. The method of Clause 11, wherein (b) raising the pH of thedigested ECM comprises adding a base or an isotonic buffer to raise thepH of the digested ECM.

Clause 13. The method of Clause 10 or Clause 11, wherein the acidprotease is pepsin, trypsin or a combination thereof.

Clause 14. The method of any one of Clauses 1-13, wherein the ECMhydrogel is maintained at or below 25° C. prior to administration to thesubject.

Clause 15. The method of any one of Clauses 1-14, wherein the subjecthas Barrett's esophagus or is at risk of Barrett's esophagus.

Clause 16. The method of Clause 15, wherein the method inhibits thedevelopment of esophageal neoplasia in the subject.

Clause 17. The method of any one of Clauses 1-16, wherein the ECMhydrogel restores the epithelial barrier in the esophagus of thesubject.

Clause 18. The method of any one of Clauses 1-17, wherein the ECMhydrogel increases chemotaxis of both epithelial cells and/or stem cellsto the site of injury in the esophagus of the subject.

Clause 19. The method of any one of Clauses 1-18, wherein the ECMhydrogel reduces esophageal stricture in the subject.

Clause 20. A composition comprising an extracellular matrix (ECM)hydrogel, wherein the ECM hydrogel has the following characteristics: a)a time to 50% gelation of less than ten minutes at about 37° C.; b) aflow viscosity sufficient for injection into the esophagus; and astiffness of i) about 10 to about 300 Pascal (Pa), ii) about 10 to about450 Pa; iii) about 10 to about 600 Pa, iv) about 5 to about 1,000 Pa, v)about 10 to 1,000 Pa, or vi) about 10 to about 70 Pa, and wherein thecomposition is formulated for administration to the esophagus.

Clause 21. The composition of Clause 20, wherein the time to 50%gelation is a) about 3 to about 30 minutes; b) about 4 to about 10minutes; or c) is about 3 to about 10 minutes, at about 37° C.

Clause 22. The composition of Clause 20 or Clause 21, wherein thehydrogel has a stiffness of about 10 to about 70 Pa.

Clause 23. The composition of any one of Clauses 20-22, comprising about2 mg/ml to about 16 mg/ml of the ECM hydrogel.

Clause 24. The composition of any one of Clauses 21-23, wherein the ECMhydrogel is produced by: (a) solubilizing decellularized extracellularmatrix (ECM) by digestion of esophageal tissue with an acid protease inan acidic solution to produce digested esophageal ECM; (b) raising thepH of the digested esophageal ECM to a pH between 7.2 and 7.8 to producea neutralized digest solution; and (c) diluting the digested esophagealECM to a concentration of about 8 mg/ml to about 12 mg/ml of the ECMhydrogel.

Clause 25. The composition of Clause 24, wherein (b) raising the pH ofthe digested ECM comprises adding a base or an isotonic buffer to raisethe pH of the digested esophageal ECM

Clause 26. The composition of Clause 24 or Clause 25, wherein the acidprotease is pepsin, trypsin or a combination thereof.

Clause 27. The composition of any one of Clauses 20-26, wherein the ECMhydrogel is maintained at or below 25° C.

Clause 28. The composition of any one of Clauses 20-27, for use ininhibiting esophageal inflammation in a subject.

Clause 29. The composition of any one of Clauses 20-27, for use inrestoring an epithelial barrier in an esophagus of a subject.

Clause 30. The composition of Clause 28 or Clause 29, wherein thesubject has Barrett's esophagus.

Clause 31. The composition of any one of Clauses 20-27, for reducingesophageal stricture in a subject.

Clause 32. A kit comprising a) a container, wherein the containercomprises the composition of any one of Clauses 20-31, or a lyophilizedform thereof, and b) instructions for using the composition.

Clause 33. A composition for use in any one of the methods of Clauses1-19.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES

Mucosal inflammation, or mucositis, is an inflammatory conditioncharacterized by swelling, irritation, and discomfort of the mucosallinings of the gastrointestinal tract. Mucositis can result in erosionsor ulcers, which can be present throughout the gastrointestinal tract.As an inflammation of the mucosal lining, which often involves infectionand/or ulceration, mucositis is a serious and often painful condition.It is disclosed herein that extracellular matrix (ECM) hydrogels are apotential therapeutic for treating mucosal inflammation, such asesophageal inflammation. The ECM hydrogel can provide a protectivebarrier from continued insult to the mucosa, promote an environment thatis anti-inflammatory, and/or facilitates repair of damaged and inflamedmucosa.

Example 1 Viscoelastic Properties of Hydrogels

Rheology was performed on homologous esophageal ECM (eECM) hydrogels fora range of ECM concentrations (4-16 mg/mL). Samples were placed on arheometer at 10° C., a temperature well below gelation, and a steadystate flow test (shear rate 0.1 -1000 1/s) was performed to determinethe viscosity profile of the ECM pre-gels (FIG. 1).

At each shear rate, viscosity increases with increasing ECMconcentration, and the ECM pre-gels are shear-thinning (viscositydecreases with increasing shear rate). Shear-thinning is a good propertyfor an ECM pre-gel that may be injected through a catheter where it canexperience a range of shear rates of 10-1000 1/s. Further evidence ofinjectability was obtained in videos, which showed ECM pre-gels(Esophageal ECM and UBM, 8-12 mg/mL tested dyed in blue) are allinjectable through an oral gavage (5 fr size, ˜15.9 G). Temperature wasthen rapidly raised to 37° C. to induce gelation, and a time sweep (0.5%oscillatory strain) was performed at 37° C. to measure gel stiffness(FIG. 2) and gelation time (FIG. 3). FIG. 2 shows that the storagemodulus (G′) or “stiffness” of the formed ECM hydrogel increases withincreasing ECM concentration. A similar trend was observed for the lossmodulus (G″), or the viscous component of the formed ECM hydrogel. Thetime to 50% gelation was measured during the time sweep test (FIG. 3).eECM shows concentration dependent gelation times i.e., gelation timedecreased with increasing ECM concentration.

Rheology was performed on two heterologous ECM hydrogels: Urinarybladder matrix ECM (UBM) and Dermal ECM (MIRM5). Viscoelastic propertiesare shown in comparison to the homologous esophageal ECM hydrogel (eECM)in FIGS. 4-6.

4 mg/mL 8 mg/mL 12 mg/mL 16 mg/mL Mean (Pa) SD (Pa) N Mean (Pa) SD (Pa)N Mean (Pa) SD (Pa) N Mean (Pa) SD (Pa) N eECM Max G′ 5.74 0.73 3 39.6443.18 3 56.95 66.72 3 205.20 58.98 3 UBM Max G′ 53.49 5.29 3 87.36 18.643 129.70 16.46 3 308.63 91.54 3 dECM Max G′ 0.42 0.57 3 3.26 1.17 335.16 33.44 3 59.18 19.64 3

A steady shear test was similarly performed as described for FIG. 1.Dermal ECM (FIG. 4B) and UBM (FIG. 4C) show a concentration-dependentincrease in viscosity with increasing ECM concentration andshear-thinning profiles of the ECM hydrogels i.e., viscosity decreaseswith increasing shear rate for each ECM concentration. The viscosityranges were distinctive for each tissue type.

UBM ECM (FIG. 5C) showed increasing storage modulus (stiffness) withincreasing ECM concentration similarly as esophageal ECM (FIG. 5A).Dermal ECM (FIG. 5B) did not form a hydrogel at the low concentration of4 mg/mL, demonstrating that not all ECM hydrogels derived from differenttissue sources behave similarly. The stiffness ranges of the three ECMhydrogels was distinctive.

Dermal ECM (FIG. 6B) showed concentration-dependent gelation times,i.e., gelation time decreased with increasing ECM concentration,similarly to esophageal ECM (FIG. 6A), while UBM showedconcentration-independent gelation times, i.e. gelation time remainedconstant for all ECM concentrations (FIG. 6C). The distinctive gelationprofiles further demonstrate the variability of the ECM hydrogel derivedfrom different tissue sources. Thus, esophageal hydrogels provide uniqueproperties, and are of use at a variety of concentrations such as, butnot limited to, from 8 mg/ml to about 12 mg/ml.

Example 2 Treating Mucosal Inflammation with an ECM Hydrogel

Mucosal inflammation, or mucositis, is an inflammatory conditioncharacterized by swelling, irritation, and discomfort of the mucosallinings of the gastrointestinal tract. Mucositis can result in ulcers,which can be present throughout the gastrointestinal tract. As aninflammation of the mucosal lining, which often involves infectionand/or ulceration, mucositis is a serious and often painful condition.

Mucositis often develops as a complication of chemo- or radiationtherapy for cancer, for example. The goal of radiation and chemotherapyin cancer treatment—to kill rapidly dividing cancer cells—also affectsepithelial cells of the mucous membranes lining regions such as thegastrointestinal tract, leading to mucositis. Exposure to radiationand/or chemotherapeutics often results in significant disruption ofcellular integrity in mucosal epithelium and the underlying connectivetissue, leading to inflammation, infection and/or ulceration at mucosalsites such as, for example, in the esophagus and other portions of theGI tract.

Extracellular matrix (ECM) hydrogels are a therapeutic for treatingmucosal inflammation. Without being bound by theory, the mechanism bywhich ECM may support mucosal healing is (1) by formation of a hydrogelthat provides a protective barrier from continued insult to the mucosa,(2) by promoting an environment that is anti-inflammatory, and/or (3) byfacilitating repair of damaged and inflamed mucosa. The properties ofesophageal hydrogels were investigated, as disclosed in Example 1.

Macrophages, when exposed to ECM hydrogels, elicit a secreted cytokineprofile that is primarily anti-inflammatory, including elevated levelsof PGE2 (FIG. 7). The level of anti-inflammatory cytokines variesdepending upon the tissue from which the ECM was derived. Esophagealhydrogels provide strong anti-inflammatory effects.

Mucosal repair requires not only inflammation reduction but also thereestablishment of an epithelial barrier and/or a physical barrier. ECMhydrogels can promote the restoration of an epithelial barrier byincreasing the chemotaxis of both epithelial cells and stem cells. Thesecreted products of macrophages exposed to ECM enhance the migration ofepithelial cells (FIG. 8A). Furthermore, ECM hydrogels directly promotethe chemotaxis of esophageal stem cells; these effects are dependentupon the source tissue from which the ECM was derived (FIG. 8B).Esophageal stem cells migrated preferentially towards esophageal ECM andsmall intestine ECM. FIG. 9 shows exemplary treatment of Barrett'sesophagus.

FIG. 10A shows the effects forty minutes after oral administration, thehydrogel remained coating the mucosa despite normal swallowing and couldbe identified. This was performed to test the mucoadhesiveness of thegel in-vivo to confirm that oral swallowing was effective. As shown inFIG. 10B, using a catheter and endoscope, the hydrogel was delivered toa specified location within the esophagus (in this case in the shape ofa ring). Thus, the hydrogel was delivered to a specific topical locationwithin the esophagus.

FIG. 11 also shows the effects of hydrogel treatment. The top row is theresult of constant reflux for at least three months in the animals, withesophageal inflammation. The three dogs to the left were treated withOmeprazole (a proton pump inhibitor) and the esophageal ECM hydrogel.The last animal (right panel) was only treated with Omeprazole. After 30days of treatment, improvement in esophageal inflammation can be seen inthe three animals that received ECM hydrogel. The animal that onlyreceived Omeprazole showed no improvement (gray box, right panels).

FIGS. 12A-12B show an evaluation of the safety of the ECM hydrogel. Theanimals underwent a procedure that increased the acid reflux to createesophagitis, and subsequently create Barrett's Esophagus. None of theeight animals lost weight after surgery to induce BE nor duringtreatment with the hydrogel (FIG. 12A). Physiological parameters wereanalyzed. After 30 days of twice daily administration of the hydrogel,the animal's physiologic parameters were stable and did not go out ofthe normal range (FIG. 12B).

In the animal model before treatment with ECM, both a dog that would betreated with the ECM hydrogel and dog that would not be treated with theECM hydrogel developed columnar metaplasia (FIGS. 13A and 13B). Aftertreatment with ECM+Omeprazole the treated dog improved and no columnarmetaplasia was seen (FIG. 13C). In the control animal, not treated withthe ECM hydrogel, there was columnar metaplasia with small patches ofsquamous epithelium at the same area (FIG. 13D).

Example 3 Treating Stricture with an ECM Hydrogel Material and Methods

A full circumferential mucosal resection of 5 cm of longitudinal widthwas performed using a combination of EMR and ESD techniques (Nieponice,2009, 18657808) in two mongrel dogs. Evaluated treatments were twicedaily administration of UBM hydrogel and an untreated control. Endoscopywas performed if animals presented any clinical signs of stricture orhad reached 1 month after the procedure. According the endoscopicfindings on the animals, a dilation was performed if possible andnecessary. Animals were euthanized if they presented severe stricture orreached the 2 month timepoint after the balloon dilation. At thenecropsy, animal tissue was measured to determine stricture and sampleswere harvested for histologic analysis. This animal model allowedmeasuring the following endpoints:

-   -   1. Endoscopic appearance of resection area    -   2. Esophageal measurements    -   3. Histologic assessment at final timepoint

Surgical Procedure & Postoperative Care

Each dog was induced with Acepromazine (0.01 mg/kg, SC) and ketamine(5-11 mg/kg), and surgical plane anesthesia maintained with 1-5%Isofluorane via endotracheal tube. After induction, the animal was movedto the surgery table and positioned inside the sterile surgical theater.Throughout the procedure and observation animals were infused with 2ml/kg/h of lactated Ringer's solution. Temperature was controlledthrough warm water recirculating heating pads placed under the animal.Physiologic parameters such as heart, respiration rate, bodytemperature, and responsiveness were monitored during the procedure.Antibiotic prophylaxis with 25 mg/kg of Cefazolin was administeredbefore starting the procedure.

The animal was placed in supine decubitus with and a Pentax EG3430Kendoscope was used to evaluate the esophagus. Distance from the mouth tothe GE junction was measured. After identifying reference points in theesophagus, the mucosa and submucosa were separated by with injection ofsaline using a Olympus Injectorforce 4 mm 23G needle. The fullcircumference of the mucosa (100%) for a length of 5 cm was removedusing the ESD and Loop EMR technique. The ESD technique was done byinjecting fluid or ECM into the mucosa/submucosa to separate the mucosafrom the submucosa then using an endoscopic TT knife to cut the area. Toperform EMR, a Cook Duette Kit with a ligation band was used. The mucosawas then excised with the use of a snare. The area of resection wasdemarked using Spot Endoscopic Marker.

After the mucosa was removed, during the procedure 50 mL of 12 mg/mL UBMhydrogel were delivered and applied to area of excision using a MILAEDC190 Endoscopic delivery catheter. The animal was maintained underanesthesia for 5 mins to allow gelation of the hydrogel. After theprocedure animals were recovered and placed in observation.

Following the surgical procedure and cessation of inhalation anesthesia,animals were continually monitored for 24 hours. Body temperature wasdetermined and recorded every 12 hours. The animals were kept warm anddry to prevent hypothermia and was rotated once per half-hour until theymaintained a sternal position.

Dogs were held in single housing with other animals in the room untilanimals were stable, and then placed in normal living facilities.Buprenorphine (0.005-0.01 mg/kg IM or IV, q12h), was administeredfollowing each surgical procedure for 5 days for pain and was continuedif signs of pain were present and cephalexin (35 mg/kg q12) for 5 days.

After the procedure and until the end of the study, animals weremonitored for signs of esophageal stenosis like decrease in foodconsumption, loss of body weight, and for signs of distress, asdetermined by increases in breathing patterns, vocal expression, emeticepisodes or difficulty swallowing food and/or decreased activity. Ifthese signs were present, animals were evaluated with a contrastesophagogram and/or endoscopy.

Endoscopic Monitoring & Balloon Dilation

One month after the initial surgical procedure animals underwent aendoscopy procedure if any clinical signs of stricture were present.Additionally, all animals underwent an endoscopic procedure prior toeuthanasia. Anesthesia was induced with acepromazine (0.1-0.5 mg/kg) andmaintained on isoflurane (1-5%) to perform the endoscopy.

If the animal was diagnosed with a mild or moderate stricture during theendoscopy, a balloon dilation was performed. To perform the dilationprocedure an Olympus 20 mm balloon dilator was used. Under endoscopicguidance, the balloon was inflated until moderate or significant amountof resistance could be identified with approximately 10 mL of 0.9% NaClsterile and was kept inflated for 30-60 seconds. After dilation, 50 mLof ECM was immediately applied to the injured area using a MILA EDC 190catheter and left to gel for 5 minutes. After the procedure animals werenot given access to food or water for at least an hour.

ECM Delivery

ECM was delivered orally to the animals with the use of a 60 mlcathether tip syringe at 15° C. 50 mL were delivered twice daily fromday 0 until completion of the study. Animals were not allowed to eat ordrink for an hour after each delivery of the hydrogel.

Necropsy

At the moment of necropsy an endoscopy was performed as previouslydescribed. Euthanasia was performed by administering PentobarbitalSodium IV (390 mg/kg BW) under anesthesia. After death was confirmed,the esophagus was harvested maintaining the same dimensions it had inthe body. Measurements of the esophagus were taken 0.5 cm apart andrecorded.

The ECM hydrogel treated stricture. As shown in FIG. 14, in a model offull circumferential resection of the mucosa (Different from theBarrett's esophagus model), animals were treated for up to 81 days withECM hydrogel. A control animal had a severe untreatable stricture andwas euthanized after 14 days. An ECM treated animal had esophagealstenosis at 21 days which was dilated. Two months after dilation, theanimal was sacrificed and esophageal measurements were taken. The ECMtreated animal had a wider internal circumference and a lower decreasein circumference when compared to the control.

As shown in FIG. 15, a control animal at 14 days showed disorganizedcollagen deposition with high cellular infiltration and erosion at thecenter where the mucosal resection was created. As shown in FIG. 16,treated animals showed re-epithelialization at the center of the defectwith lower cellular infiltration and a more organized and dense collagendeposition.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

1. A method for inhibiting esophageal inflammation or reducingesophageal stricture in a subject, comprising administering to theesophagus of the subject with esophageal inflammation a therapeuticallyeffective amount of an extracellular matrix (ECM) hydrogel, wherein theECM hydrogel has the following characteristics: a) a time to 50%gelation of less than 30 minutes at a temperature of about 37° C.; b) aflow viscosity suitable for infusion into the esophagus; and c) astiffness of about 10 to about 300 Pascal (Pa); thereby inhibitingesophageal inflammation or reducing esophageal stricture in the subject.2. The method of claim 1, wherein the time to 50% gelation is about 3 toabout 30 minutes at about 37° C.
 3. The method of claim 1, wherein thetime to 50% gelation is about 3 to about 10 minutes at about 37° C. 4.The method of claim 2, wherein the time to 50% gelation is about 4 toabout 10 minutes.
 5. The method of claim 1, wherein the flow viscosityis about 1 to about 40 Pa*s at a shear rate of about 0.1/s and is about0.01 to about 0.2 Pa*s at a shear rate of 1000/s.
 6. The method of claim1, wherein the flow viscosity is about 0.1 to about 25 Pa*s at a shearrate of 1/s, and is about 0.02 to about 0.8 Pa*s at a shear rate ofabout 100/s.
 7. The method of claim 1, wherein the ECM hydrogel has astiffness of 10-70 Pa.
 8. The method of claim 1, wherein the ECMhydrogel is an esophageal ECM hydrogel.
 9. The method of claim 1,wherein the ECM concentration in the ECM hydrogel is 2 mg/ml to about 16mg/ml.
 10. The method of claim 1, wherein the ECM hydrogel isadministered orally, endoscopically or via a catheter to the subject.11. The method of claim 1, wherein the ECM hydrogel is produced by (a)solubilizing decellularized extracellular matrix (ECM) by digestion oftissue with an acid protease in an acidic solution to produce digestedesophageal ECM; and (b) raising the pH of the digested esophageal ECM toa pH between 7.2 and 7.8 to produce a neutralized digest solution. 12.The method of claim 11, wherein (b) raising the pH of the digested ECMcomprises adding a base or an isotonic buffer to raise the pH of thedigested ECM.
 13. The method of claim 10, wherein the acid protease ispepsin, trypsin or a combination thereof.
 14. The method of claim 1,wherein the ECM hydrogel is maintained at or below 25° C. prior toadministration to the subject.
 15. The method of claim 1, wherein thesubject has Barrett's esophagus or is at risk of Barrett's esophagus.16. The method of claim 15, wherein the method inhibits the developmentof esophageal neoplasia in the subject.
 17. The method of claim 1,wherein the ECM hydrogel a) restores the epithelial barrier in theesophagus of the subject; b) increases chemotaxis of both epithelialcells and/or stem cells to the site of injury in the esophagus of thesubject; and/or c) reduces esophageal stricture in the subject. 18-19.(canceled)
 20. A composition comprising an extracellular matrix (ECM)hydrogel, wherein the ECM hydrogel has the following characteristics: a)a time to 50% gelation of less than ten minutes at about 37° C.; b) aflow viscosity sufficient for injection into the esophagus; c) astiffness of about 10 to about 300 Pascal (Pa); and d) the hydrogel isan esophageal hydrogel; and wherein the composition is formulated foradministration to the esophagus. 21-31. (canceled)
 32. A kit comprisinga) a container, wherein the container comprises the composition of claim20, or a lyophilized form thereof, and b) instructions for using thecomposition.